Under hypocalcemic conditions a unique substance is eluted from the in situ perfused rat liver which, when partially purified, noncompetitively decreases adenylate cyclase activation by parathyroid hormone, epinephrine and glucagon in several tissues and in several species; higher concentrations of this substance decrease basal, and flouride activation of enzyme also. The inhibitor is a peptide, MW equals 1,000, which is heat and acid stable, alkali labile, and is destroyed by several proteolytic enzymes. In the proposed studies we will attempt to isolate, purify, and chemically characterize this substance, and study its distribution in the cell, and in various tissues and body fluids under a variety of physiological conditions. In addition we intend to produce a radioactively labeled analog of this compound, and raise antibodies against it. This will make the development of a radioimmunoassay for this substance possible. The role of this inhibitor in pathological conditions related to cyclic AMP mediated hormone responsiveness in hypocalcemic states will be investigated. Utilizing infusion studies in rats, additional studies will be done to determine the effects of the inhibitor in vivo on such cyclic AMP-triggered processes as glucagon-induced glycogenolysis, glucagon and catecholamine-induced lipolysis; glucagon-induced tachycardia, and hormone-mediated changes in serum and urinary cyclic AMP levels. We intend to investigate the effect of the inhibitor in vitro on a number of enzymes that have been shown to be involved either in the breakdown of cyclic nucleotides, or whose activity is altered by cyclic AMP or cyclic GMP. These include phosphodieterases, protein kinases, guanylate cyclase, phosphorylase, glycogen synthase, phosphatase and ATPase. The levels of cyclic AMP and the inhibitor will be measured in the perfused rat liver preparations in response to hormones and exogenously administered dibutyryl cyclic AMP in normocalcemic and hypocalcemic conditions. The role of the inhibitor in hormone refractoriness will be investigated systematically.